The goal of the work is to advance our original studies on developing the scientific principles required for RNAi-based clinical therapeutics. Our plan is to provide a platform for safe and effective transcription-based RNA interference (RNAi) into vital organs such as the liver. We are specifically interested in developing a therapeutic approach for Hepatitis C Virus Infection (HCV) for which there is still an unmet clinical need. Until recently, research in this area has been hampered by the lack of ability to study replication from an intact viral infection in cell culture and the paucity of animal models. As a result, HCV replicons have been the primary means to study viral replication. We have discovered that some RNAi targets are effective against replicons but relatively inert against bona-fide viral infection that shares identical target sequences. We plan to re-evaluate various regions of the HCV genome for RNAi knockdown based on our new models and information regarding shRNA-vector biology. In addition, we will attempt to definitively evaluate the effectiveness of targeting the HCV minus strand because it is produced in much lower amounts in infected cells. We will determine if targeting both strands simultaneously will provide better protection and lessen the likelihood of quasi-species formation. We plan to use cell culture and humanized mouse liver models with our newly developed recombinant AAV vectors and determine the validity of RNAi as a means to protect against HCV exposure as well as elimination of the virus after establishment of a sustained infection. By the end of the granting period, we believe we will be able to provide important insights into the parameters required for effective vector-based RNAi in vivo and have a robust clinically relevant means of treating HCV infection as a single therapy and/or in combination with other non-nucleic acid based therapeutics.